The present invention relates to recombinant DNA that encodes the AcuI restriction endonuclease (AcuI endonuclease or AcuIR) as well as the AcuI methyltransferase (AcuI methylase or M.AcuI), and expression of AcuI endonuclease and methylase in E. coli cells containing the recombinant DNA. AcuI is an isoschizomer of Eco57I (MBI Fermentas (Vilnius, Lithuania) product #ER0341).
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind to particular sequences of nucleotides (the ‘recognition sequence’) along DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. At least two hundred and forty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts et al., Nucl. Acids Res. 31:418-420 (2002)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′RG/GNCCY3′ and 5′CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.
A second component of bacterial/viral restriction-modification (R-M) systems is the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group to produce C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine. Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiable foreign DNA, is susceptible to restriction endonuclease recognition and cleavage. During and after DNA replication, usually hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction endonuclease.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, (i.e. populations of clones derived by ‘shotgun’ procedures) when they occur at frequencies as low as 10−3 to 10−4. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985); Tsp45I: Wayne et al. Gene 202:83-88 (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and PstI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).
A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on an indicator strain of E. coli containing the dinD::IacZ fusion (U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signal following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5-cytosine methylases, N4-cytosine methylases, and N6-adenine methylases). N4-cytosine and N6-adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632 (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer DNA sites resistant to restriction digestion. For example, Dcm methylase modification of 5′ CCWGG 3′ (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucleotide of the NotI site (5′ GCGGCCGC 3′) and make it refractory to NotI digestion (New England Biolabs' (Beverly, Mass.) catalog, 2002-03, page 252). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Type II methylase genes have been found in many sequenced bacterial genomes (GenBank, http://www.ncbi.nlm.nih.gov; and Rebase®, http://rebase.neb.com/rebase). Direct cloning and over-expression of ORFs adjacent to methylase genes yielded restriction enzymes with novel specificities (Kong et al. Nucl. Acids Res. 28:3216-3223 (2000)). Thus microbial genome mining emerged as a new way of screening/cloning new type II restriction enzymes and methylases and their isoschizomers.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant DNA molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes and methylases. Such over-expression strains should also simplify the task of enzyme purification.
AcuI recognizes the double-stranded DNA sequence 5′CTGAAG3′ (or 5′CTTCAG3′ bottom strand) and cleaves 16/14 bases downstream of its recognition sequence to generate a 2-base 3′ cohesive end. AcuI is classified as a type IIs restriction enzyme since it cleaves DNA downstream from its recognition site. In addition, AcuI was expected to be a type IIG enzyme as it is an isoschizomer of Eco57I, the first such restriction enzyme to be identified (Janulaitis et al. Nucl. Acids. Res. 20:6043-6049 (1992)). Type IIG restriction endonucleases are distinguished by the fact that they possess both restriction and modification activity in one polypeptide chain (Pingoud and Jeltsch, Nucl. Acids Res. 29:3705-3727 (2001)). Therefore, when such an enzyme is employed in vitro to digest DNA, two competing activities are at work. If the modification (methylation) activity is significant, some of the substrate recognition sites may become modified before the endonuclease function is complete. This outcome is clearly apparent when using Eco57I to cleave lambda DNA, for example. (see FIG. 1). In contrast, when native purified AcuI is used to cleave the same substrate, complete digestion is observed. Therefore, an attempt was made to clone the AcuI restriction-modification system into E. coli in order to over-express and purify commercial quantities of the AcuI restriction endonuclease.